Glioblastoma is the most common and most malignant of human brain tumors. Recent therapeutic advances have improved control of primary tumors, with a significant fraction of tumors responding to initial therapies. Unfortunately, all glioblastomas recur and lead to patient death. For this reason, the present proposal focuses on the crucial issue of recurrent glioblastoma-with studies extending from biomarker generation and evaluation, to understanding mechanisms of therapeutic resistance, to exploration of novel therapeutic compounds. Over the past 15 years, studies supported by this grant have clarified the molecular genetic basis of glioblastomas and other malignant gliomas and have shown that the treatment of such tumors can be guided by molecular markers; in particular, the now widespread clinical use of 1p/19q testing in oligodendrogliomas emerged from studies performed under this grant. Preliminary data gathered during the last funding period have shown: glioblastomas become resistant to the effects of the alkylating agent used to treat all glioblastomas, temozolomide (TMZ), through a mechanism associated with inactivation of MSH6; MSH6-deficient glioblastomas grow more rapidly during TMZ therapy; and in vitro resources exist to evaluate defects in the TMZ-induced DNA damage response and therapeutic resistance. To address this critical issue, we propose three Specific Aims: 1) To define the spectrum of DNA repair defects that arise after TMZ therapy in glioblastoma, and to generate relevant biomarkers relating these events to clinical outcome; 2) To define the mechanism whereby deficiencies in DNA damage responses enable therapeutic resistance; and 3) To define pathways that could be targeted to overcome therapeutic resistance due to defects in DNA damage response. The proposed approach will therefore extend our prior clinically relevant biomarker work to the particularly vexing problem of recurrent glioblastoma, a tumor that confounds current treatment attempts. Importantly, through continuing the work supported by this grant over the past three funding cycles, we aim to generate biomarkers and elucidate mechanisms of therapeutic resistance that could translate in the short term to changes in current diagnostic and therapeutic approaches and, in the long term, to the development of novel targeted therapies.